In North America, there are over 6 million pregnancies annually. Rhesus hemolytic (Rh) disease of the newborn is a serious alloimmune condition, where fetal red blood cells (RBCs) are destroyed by the maternal immune system through the placenta. During pregnancy or child birth, a small number of fetus's RBCs can enter the mother's circulation. If the mother is Rh negative and the fetus is Rh positive, the mother's body produces antibodies (IgG) against the Rh antigen. This process is termed sensitizing. Most frequently, sensitizing occurs during child birth (about 85% of sensitized cases), but fetal blood may pass into the maternal circulation earlier during the pregnancy (about 15% of sensitized cases) (see J. M. Bowman and J. M. Pollock, “Rh-Immunization during Pregnancy and Grandmother Theory,” Journal of Pediatrics, vol. 93, pp. 313-314, 1978). During this or subsequent pregnancies the IgG is able to pass through the placenta into the fetus and consequently cause the destruction of the Rh positive fetal RBCs.
In Caucasian populations about 1 in 10 of all pregnancies are of a Rh negative woman with a Rh positive baby, of which 13% Rh negative mothers are sensitized (see A. S. Prasad, Ed., Acquired hemolytic anemias. In: Bick R L, ed. Hematology: Clinical and Laboratory Practice. St. Louis: Mosby-Yearbook, Inc., 1993). Besides the resulting anemia, fetal-maternal hemorrhage may have devastating consequences for the fetus such as neurologic injury, stillbirth, or neonatal death (B. J. Wylie and M. E. D'Alton, “Fetomaternal hemorrhage” Obstet. Gynecol, Vol. 115, pp. 1039-51, 2010). Many babies who managed to survive would be severely ill. The diagnosis and treatment require quantifying fetal-maternal hemorrhage (i.e., the amount of fetal blood that has passed into the maternal circulation), which is usually performed on Rhesus-negative mothers to determine the required dose of the drug, Rho(D) immune globulin (Rhlg) to inhibit the formation of Rh antibodies in the mother and prevent Rh disease in future Rh-positive children.
Besides Rh diseases, fetal-maternal hemorrhage can also result from the loss of integrity of the normal physiological barrier between the fetal and maternal circulation, which must be quantified in pregnancy care for prompt treatment (e.g., blood transfusion) (C. J. Chen, S. N. Cheng, C M. Lee, F. W. Chang, C. C. Wu, and Y. S. Yuh, “Fetomaternal hemorrhage,” J. Med. Sci, Vol. 23, pp. 231-34, 2003). The standard clinical method of quantifying fetal-maternal hemorrhage is the Kleihauer-Betke. (KB) test (Z. Y. Wang, J. W. Shi, Y. L. Zhou, and C. G. Ruan, “Detection of red blood cell-bound immunoglobulin G by flow cytometry and its application in the diagnosis of autoimmune hemolytic anemia,” International Journal of Hematology, vol. 73, pp. 188-193, 2001). The test takes advantage of the differential resistance of fetal and maternal hemoglobin to acid (fetal hemoglobin is significantly more resistant). A standard blood smear is prepared from the mother's blood (1:1,000 dilution with PBS). After drying, staining and incubating, the blood smear slides are counted under a microscope by certified technologists. Since the fetal hemoglobin is resistant to the citrate buffer, the resulting bright-pink cells are classified as fetal cells. The percentage of fetal-maternal hemorrhage can then be calculated.
The critical component of the KB test is the counting of fetal and maternal RBCs. This counting is presently done manually by trained technologists who look into the eyepieces of a microscope and count a minimum of 2,000 cells. Manual counting takes about 15 minutes and inherently suffers from inconsistency and unreliability.
Although the term ‘automated detection of fetal RBCs’ was mentioned in the literature, there have been no reported approaches for automated counting of fetal and maternal RBCs, and there have been no reported approaches for distinguishing these two types of cells. In the work reported in the literature (see D. M. V Pelikan, W. E. Mesker, S. a Scherjon, H. H. H. Kanhai, and H. S. Tanke, “improvement of the Kleihauer-Betke test by automated detection of fetal erythrocytes in maternal blood,” Cytometry. Part B, Clinical cytometry, vol. 54, no. 1, pp. 1-9, July 2003 and see D. M. Pelikan, S. a Scherjon, W. E. Mesker, G. M. de Groot-Swings, G. G. Brouwer-Mandema, H. J. Tanke, and H. H. Kanhai, “Quantification of fetomaternal hemorrhage: a comparative study of the manual and automated microscopic Kleihauer-Betke tests and flow cytometry in clinical samples,” American journal obstetrics and gynecology, vol. 191 no. 2, pp. 551-7, August 2004), automation in these works refers to the use of commercial motorized stage to capture cell images. Images were captured first with a green filter, and images were captured again in a second scan with a red filter. Hence, image capturing required the use of a microscope equipped with a green absorption filter and a red absorption filter. Fetal RBCs were distinguished from maternal RBCs based on intensity and distribution patterns of staining by a technologist. The total cell number on a KB slide was also manually estimated.
Image processing methods have been developed for counting other types of cells, but not for counting RBCs. In one study (see N. Bandekar, A. Wong, D. Clausi and M. Gorbet, “A novel approach to automated cell counting for studying human corneal epithelial cells,” Intn'l Conf. IEEE EMBS, Boston, pp. 5997-6000, 2011), cell counting was done via non-maximum suppression and seeded region growth to segment the clustering corneal epithelial cells. Nevertheless, only grey-level and spatial information were applied in this algorithm making it difficult to detect ghost cells and segment those clustered cells. In another study, the Hough circle transform was used to automatically count cell colonies in a culturing flask (see J. M. Bewes et al., “Automated cell colony counting and analysis using the circular Hough image transform algorithm (CHiTA),” Phys. Med. Biol, Vol. 53, pp. 5991-6008, 2008). Intensity gradient and circular Hough transform were used to discriminate the colony edges and detect cells. It was useful for quantifying low concentrations of cell colonies while under-segmentations are inevitable on those severely overlapping cells. Furthermore, contaminants cannot be excluded from the counting result.
Prior art patents include U.S. Pat. No. 6,341,180, U.S. Pat. No. 7,327,901 and U.S. Pat. No. 7,835,077 assigned to CellVision; and patent publications US2011/0170760 to Nextslide Imaging, US2010/0189338 to Nexcelom Bioscience, and US2012/0314092 to Bio-Rad Laboratories, Inc. The following three are considered relevant to the present invention.
“Network Image Review in Clinical Hematology”, US2011/0170760 by Nextslide Imaging, disclosed a method for setting up an internet-based database for cell digital image analysis. Users upload cell images via the internet, and the images are processed on a remote server. No cell counting methods are disclosed.
U.S. Pat. No. 7,835,077, by CellaVision AB, “Microscope system comprising arrangement for positioning of a platform”, describes a flexture system to adjust a specimen's vertical position for keeping the specimen within focus.
“Cell Counting System and Methods”, as disclosed in US2012/0314092 by Bio-Rad Laboratories, Inc., introduced a system for counting cell numbers using digital images of a sample. The sample holder holding a set of cells is inserted into the slot of the system and the motion mechanism can change the relative positions of the sample holder automatically. Not more than three images are captured during the rotation and translation of the sample holder. The majority of areas on the sample are omitted making counting accuracy undependable. Furthermore, this system is applicable only to the testing of samples having low cell concentrations.
Accordingly, the prior art is silent on automated and sufficiently qualitative method or system for counting and distinguishing between maternal and fetal red blood cells. It is an object of the invention described below to provide a method and/or system that addresses at least one of the aforementioned problems with the prior art.